Blood consists of cells suspended in a protein rich fluid called plasma. There are three major groups of cells in blood: red cells, white cells and platelets. Platelets can, when they come into contact with certain materials and chemicals (especially those released from damaged cells), undergo a process known as the aggregation-adhesion reaction. When they aggregate, platelets change from a discoid shape to a more spherical form, extend long processes known as pseudopodia and become sticky. As a result, the platelets stick to one another and to the damaged tissue, thus plugging gaps or holes in the blood vessel wall. Although the primary response of platelets is to aggregate, a secondary release reaction may also occur, during which platelets release materials which accelerate the clotting process.
The phenomenon of aggregation is a widely studied property of platelets. It is of interest not only for scientific reasons since, inter alia, platelets make an ideal test system for examining cellular mechanisms and drug action, but also has diagnostic significance since there are many conditions in which platelet function is abnormal, and screening of platelet function is a common hematological test. Instruments used to analyze aggregation are known as aggregometers.
An early development in the aggregometer art was the Born aggregometer. The Born aggregometer analyzes aggregation response in samples of platelet-rich plasma (PRP) by measuring light transmission through the sample. In untreated PRP, the majority of the light is scattered by the platelets and transmission is minimal. On the other hand, when an aggregating agent is added to the stirred sample, the platelets clump together and light transmission increases. One serious drawback of the Born aggregometer, and conventional optical aggregometers in general, is the necessity to first separate the blood by centrifugation to obtain samples of PRP and platelet poor plasma (PPP).
Another type of device for analyzing aggregation is the to membrane capacitance aggregometer, in which a change in capacitance between two electrodes resulting from platelet aggregation is measured. However, measurement of capacitance, or even change in capacitance in the capacitance range in question, is difficult, and such an apparatus tends to be prone to drift and disturbance by outside influences.
U.S. Pat. No. 4,319,194 to Cardinal et al. discloses an aggregometer which analyzes platelet aggregation by passing a very small electric current between two electrodes immersed in a sample of blood or PRP and measuring the electrical impedance between the electrodes. During initial contact with the blood or PRP, the electrodes become coated with a monolayer of platelets. When an aggregating agent is added, platelets gradually accumulate on the monolayer coating, increasing the impedance between the electrodes. The change in impedance is recorded as a function of time.
Cardinal et al. eliminated the need to centrifuge blood to obtain PRP and PPP for use in measuring aggregation of platelets optically. The ability to speed-up testing, reduce labor costs, and test the platelets in their natural milieu was an important advance in platelet studies. The measurement in whole blood also allows studies to be performed in cases where optical aggregation is not reliable, such as with giant platelets (Bernard-Soulier syndrome), where red cells have been lysed or where it is difficult to obtain enough blood to make PRP and PPP, such as with small animals or babies.
The aggregometer of Cardinal et al. employs round or rod-shaped wires as electrodes, failing to appreciate certain disadvantages of these wires. The wires are pliable and unless attached at both ends, there can be movement of the wires during handling and cleaning, causing inconsistent results. The shapes of the electrodes and supporting structure cause variations in the flow pattern from electrode to electrode. These variations require testing and matching of electrodes, which increase the manufacturing costs. Each electrode requires exact placement of the wires during fabrication, making the final product expensive and therefore not disposable after each test.
Cardinal et al. prefers that the electrodes comprise precious metals since base metals drift in blood/saline mixtures; however, precious metal electrodes are too expensive to be disposable. Therefore, the electrode assembly must be cleaned by hand between tests, exposing the operator to contact with the sample, and thus potentially exposing the operator to diseases transmitted through the fluids contained in the sample. Since diseases such as hepatitis and AIDS can be transmitted through handling of blood products, there is an understandable reluctance on the part of medical professionals to handle blood, blood products and objects contaminated therewith.
U.S. Pat. No. 4,591,793 to Freilich addresses at least some of the foregoing problems by substituting for the wire electrodes a conductive ink printed on a plastic nonreactive base. This device is less expensive than the Cardinal et al. device and is disposable after each test; however, there are disadvantages to the Freilich device as well. The platelets have difficulty adhering to the exposed conductive surface of the Freilich device, probably due to the surface being thin. Sometimes the aggregated platelets break off the surface, causing a sudden change in impedance. Although the Freilich device is inexpensive to manufacture, the measurements returned by the device are inconsistent and not reproducible.
Accordingly, there is a need for a disposable, but accurate and reliable, platelet aggregation measuring system in which the items that contact the sample, such as the cuvette, the electrode and the stirring agitator, are discarded after a single use, particularly in clinical applications. With a single-use disposable system, it is not necessary to retrieve, cleanse and re-use the electrode assembly and/or other items such as the stir bar that have been in contact with the blood.